Quantitative determination of the neutral glycosyl ceramides in human blood

Abstract

A method is described for the qualitative and quantitative estimation of four neutral glycosyl ceramides from human plasma and erythrocytes. Total lipids extracted from 50 ml of plasma or packed erythrocytes were separated by silicic acid chromatography into neutral lipids, a fraction of mixed glycolipids that was eluted with acetone-methanol 9:1, and phospholipids. After mild alkali-catalyzed methanolysis to remove contaminants from the crude fraction of glycolipids, individual glycosyl ceramides were isolated by preparative thin-layer chromatography. The oligosaccharide portions of these lipids were characterized by cleavage with methanolic hydrogen chloride and gas chromatography of the O-trimethylsilyl methyl glycosides. It was possible to study the composition of the carbohydrate and sphingolipid base fractions in the same gas chromatographic analysis. With mannitol as an internal standard for gas chromatographic estimation of glucose, concentrations of each of the glycosyl ceramides were determined with a precision of about 10%. Recoveries of the lipids from plasma varied with the complexity of the oligosaccharide moiety and ranged from 94% with glucosyl ceramide to 71% with globoside. Concentrations of the four glycosyl ceramides in plasma and in erythrocytes were determined for samples from young, healthy males. Amounts of glycolipid as low as 0.1 micromole can be determined conveniently by this procedure.